Method and system for multi-view episcopic selective plane illumination microscope
A method of performing imaging includes operating a light sheet projection module in a first state during a first measurement process and using a first primary objective for illumination of a specimen using a light sheet and detection of a first fluorescent emission. The method also includes operating the light sheet projection module in a second state during a second measurement process and using a second primary objective for illumination of the specimen using the light sheet and detection of a second fluorescent emission.
1. A multi-view selective plane imaging microscope comprising:
an illumination source;
an image detector;
a specimen stage operable to support a specimen having a first side and a second side opposing the first side, wherein an optical axis is disposed normal to the specimen;
a first objective disposed along the optical axis on the first side of the specimen;
a second objective disposed along the optical axis on the second side of the specimen;
an angle adjustment module operable to position a light sheet at a predetermined angle with respect to the optical axis;
a light sheet projection module operable to direct the light sheet to either the first objective or the second objective; and
an orientation control module operable to modify a scan direction of the light sheet.
2. The multi-view selective plane imaging microscope of claim 1 wherein the light sheet projection module is operable to direct the light sheet to impinge on:
the first side of the specimen during collection of a first set of volumetric images; and
the second side of the specimen during collection of a second set of volumetric images.
3. The multi-view selective plane imaging microscope of claim 2 wherein the first set of volumetric images are collected using the first objective.
4. The multi-view selective plane imaging microscope of claim 2 wherein the second set of volumetric images are collected using the second objective.
5. The multi-view selective plane imaging microscope of claim 1 wherein the optical axis passes through the specimen.
6. The multi-view selective plane imaging microscope of claim 2 wherein the orientation control module is:
disposed in a first state during collection of a first volumetric image of the first set of volumetric images and in a second state during collection of a second volumetric image of the first set of volumetric images; and
disposed in the first state during collection of a first volumetric image of the second set of volumetric images and in the second state during collection of a second volumetric image of the second set of volumetric images.
7. A fluorescence imaging system comprising:
a fluorescent excitation source optically coupled to illumination optics, wherein the fluorescent excitation source and the illumination optics are operable to provide a light sheet;
a specimen stage operable to support a specimen having a first side and a second side opposing the first side;
an excitation path disposed between the illumination optics and the specimen stage;
an angle adjustment module disposed along the excitation path and operable to modify a scan angle of the light sheet;
an orientation control module disposed along the excitation path and operable to modify a scan direction of the light sheet;
a first objective disposed on the first side of the specimen;
a second objective disposed on the second side of the specimen;
a light sheet projection module disposed along the excitation path and operable to either:
project the light sheet to pass through the first objective and impinge on the first side of the specimen; or
project the light sheet to pass through the second objective and impinge on the second side of the specimen; and
an image detector optically coupled to detection optics.
8. The fluorescence imaging system of claim 7 wherein fluorescent emission from the first side of the specimen passes through the first objective.
9. The fluorescence imaging system of claim 8 wherein fluorescent emission from the second side of the specimen passes through the second objective.
10. The fluorescence imaging system of claim 9 wherein:
the fluorescent emission from the first side of the specimen propagates along at least a portion of the excitation path; or
the fluorescent emission from the second side of the specimen propagates along at least a portion of the excitation path.
11. The fluorescence imaging system of claim 7 wherein the specimen stage is characterized by a specimen plane aligned with an x-y plane, the first objective is disposed a first distance from the specimen plane measured along a positive z-axis, and the second objective is disposed at a second distance from the specimen plane measured along a negative z-axis.
12. The fluorescence imaging system of claim 11 wherein an optical axis of the first objective and an optical axis of the second objective are collinear.
13. The fluorescence imaging system of claim 7 wherein the angle adjustment module comprises a tiltable reflector.
14. The fluorescence imaging system of claim 7 wherein the angle adjustment module comprises a translatable reflector.
15. The fluorescence imaging system of claim 7 wherein the orientation control module comprises a set of moveable reflectors and a fixed reflector.
16. The fluorescence imaging system of claim 15 wherein the set of moveable reflectors are operable to:
reflect light to the fixed reflector and reflect light from the fixed reflector, respectively, in a first state; and
not reflect light to the fixed reflector or reflect light from the fixed reflector, respectively, in a second state.
17. The fluorescence imaging system of claim 7 wherein the orientation control module comprises a set of tiltable reflectors and a set of three fixed reflectors.
18. The fluorescence imaging system of claim 17 wherein the set of tiltable reflectors are operable to:
reflect light to one of the fixed reflectors in a first state; and
reflect light to a second of the fixed reflectors and reflect light from a third of the fixed reflectors, respectively, in a second state.
19. The fluorescence imaging system of claim 7 wherein the light sheet projection module comprises a tiltable reflector.